SKA1 Expression Research Articles

SKA3 overexpression predicts poor outcomes in skin cutaneous melanoma patients

BackgroundSpindle and Kinetochore Associated Complex Subunit 3 (SKA3) is a part of the SKA complex, which plays a key role in cell mitosis. Studies have shown that SKA3 was associated with cancer progression. However, its role in skin cutaneous melanoma (SKCM) remains unclear. Here, we investigated the expression level and prognostic value of SKA3 in SKCM. MethodsBased on public databases, univariate and multivariate Cox regression analyses were used to investigate the different expression of SKA3 between SKCM and normal tissues. Then, the relationship between SKA3 expression level and prognosis was assessed. PPI network and functional enrichment analysis were performed. ESTIMATE and CIBERSORT were expected to evaluate the SKA3 expression and immune status. CCK8, wound healing, transwell assays and tumor xenograft trial were performed to detect the SKA3 function in cell viability, migration and invasion of the cell lines. ResultsThe SKA3 was highly expressed in SKCM tissues. SKA3 overexpression was associated with poor survival and immune status. SKA3 knockdown inhibited cell viability, migration and invasion of SKCM cells. ConclusionSKA3 is involved in the progression of SKCM and may serve as a new prognostic biomarker and therapeutic target.

SKA3, negatively regulated by miR-128-3p, promotes the progression of non-small-cell lung cancer.

Aim: To investigate the effects of SKA3on cell proliferation and metastasis in non-small-cell lung cancer (NSCLC) and its underlying mechanism. Methods: Immunohistochemistry was employed to analyze the expression of SKA3 in NSCLC. CCK-8 assay, EdU assay, Transwell assay and flow cytometry analysis were employed to assess cell proliferation, metastatic potentialand apoptosis in vitro, respectively. A lung metastasis model was used to evaluate metastasis of NSCLC cells in vivo.A luciferase reporter gene assay was conducted to verify the targeting relationship. Results: SKA3 exhibited high expression in NSCLC tissues and cells. Overexpression of SKA3 remarkably accelerated cell proliferation and metastasis and suppressed apoptosis of NSCLC cells and promoted lung metastasis in a mousemodel. miR-128-3p repressed SKA3 expression by targeting it. Conclusion:miR-128-3p inhibited the progression of NSCLC through targeting SKA3.

Integrative Transcriptome Profiling Reveals SKA3 as a Novel Prognostic Marker in Non-Muscle Invasive Bladder Cancer.

Simple SummaryApproximately 80% of all new bladder cancer patients are diagnosed with non-muscle invasive bladder cancer. However, about 15% of them progress to muscle-invasive bladder cancer, which is associated with poor prognosis. As these diseases are highly heterogeneous, the identification of an accurate prognostic biomarker for them has been challenging. In the present study, our aims were to compare cancerous and normal adjacent bladder tissues excised from patients in various cohorts and endeavor to detect differentially expressed genes known to be upregulated exclusively in patients at high risk of bladder cancer progression. Through various molecular methods, we found that the expression level of the cell cycle-regulating SKA3 gene was markedly increased in patients with worse prognosis and high progression risk. Hence, SKA3 is a viable predictive marker for bladder cancer progression and its inhibition could constitute part of a novel therapy that improves the prognosis of this disease.Approximately 80% of all new bladder cancer patients are diagnosed with non-muscle invasive bladder cancer (NMIBC). However, approximately 15% of them progress to muscle-invasive bladder cancer (MIBC), for which prognosis is poor. The current study aimed to improve diagnostic accuracy associated with clinical outcomes in NMIBC patients. Nevertheless, it has been challenging to identify molecular biomarkers that accurately predict MIBC progression because this disease is complex and heterogeneous. Through integrative transcriptome profiling, we showed that high SKA3 expression is associated with poor clinical outcomes and MIBC progression. We performed RNA sequencing on human tumor tissues to identify candidate biomarkers in NMIBC. We then selected genes with prognostic significance by analyzing public datasets from multiple cohorts of bladder cancer patients. We found that SKA3 was associated with NMIBC pathophysiology and poor survival. We analyzed public single-cell RNA-sequencing (scRNA-seq) data for bladder cancer to dissect transcriptional tumor heterogeneity. SKA3 was expressed in an epithelial cell subpopulation expressing genes regulating the cell cycle. Knockdown experiments confirmed that SKA3 promotes bladder cancer cell proliferation by accelerating G2/M transition. Hence, SKA3 is a new prognostic marker for predicting NMIBC progression. Its inhibition could form part of a novel treatment lowering the probability of bladder cancer progression.

MiR-1207-5p suppresses laryngeal squamous cell carcinoma progression by downregulating SKA3 and inhibiting epithelial-mesenchymal transition

Laryngeal squamous cell carcinoma (LSCC) is the second most common head and neck cancer. Previously, we discovered that miR-1207-5p was downregulated in LSCC. In this study, the clinical significance, function, and mechanism of miR-1207-5p in LSCC were investigated. Downregulation of miR-1207-5p was found to be strongly linked to the malignant progression of LSCC. Functional studies revealed that miR-1207-5p upregulation suppressed LSCC cell proliferation, invasion, migration, and xenograft tumor growth. Bioinformatics analysis revealed that miR-1207-5p target genes were involved in cell cycle regulation, proliferation, adhesion, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Mechanistic studies revealed that miR-1207-5p interacts directly with the 3′ untranslated region of spindle and kinetochore associated complex subunit 3 (SKA3) and downregulates SKA3 expression. Furthermore, SKA3 was found to be overexpressed in LSCC, and its high expression was associated with tumor progression and a poor prognosis. Rescue experiments demonstrated that miR-1207-5p inhibited the malignant phenotypes of LSCC via SKA3. Furthermore, miR-1207-5p upregulation or knockdown of SKA3 inhibited the epithelial-mesenchymal transition (EMT). Collectively, miR-1207-5p inhibited LSCC malignant progression by downregulating SKA3 and preventing EMT. These findings provide new insights into the mechanism of LSCC progression, as well as new potential biomarkers and therapeutic targets for LSCC diagnosis and treatment.

SKA3 promotes glioblastoma proliferation and invasion by enhancing the activation of Wnt/β-catenin signaling via modulation of the Akt/GSK-3β axis

Spindle and kinetochore-related complex subunit 3 (SKA3) is a key modulator of the progression of multiple tumor types. However, the involvement of SKA3 in glioblastoma (GBM) has not been well studied. The current study aimed to explore the role of SKA3 expression and the potential function of the protein in GBM. Our data showed that SKA3 expression was significantly up-regulated in GBM. Functional assays demonstrated that the knockdown of SKA3 impeded the proliferation, colony formation and invasion of GBM cells, while SKA3 overexpression produced the opposite effects. Further investigation revealed that SKA3 overexpression enhanced the activation of Wnt/β-catenin signaling, which was associated with the enhanced phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β). Notably, the inhibition of Akt markedly abrogated the SKA3 overexpression-induced promotion of Wnt/β-catenin signaling in GBM cells. Further, the inhibition of Wnt/β-catenin signaling markedly abrogated the SKA3 overexpression-induced promotion of tumor growth. In addition, the knockdown of SKA3 significantly retarded tumor formation and GBM progression in vivo. In summary, these data demonstrate that SKA3 exerts promotes tumor growth in GBM by enhancing the activation of Wnt/β-catenin signaling via modulation of the Akt/GSK-3β axis. This work highlights the pivotal role of SKA3/Akt/GSK-3β/Wnt/β-catenin signaling in the progression of GBM and suggests that SKA3 is an attractive therapeutic target with potential to be used to treat GBM.

Upregulation of SKA3 enhances cell proliferation and correlates with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive types of malignancy worldwide. However, the mechanism underlying its frequent recurrence remains unclear. Studies have demonstrated that spindle and kinetochore associated complex subunit3(SKA3) is highly expressed in colorectal and prostate cancer. The present study aimed to determine whether SKA3 could be a predictive and prognostic marker for liver cancer. SKA3 expression levels inliver cancer cell lines, liver cancer tissues, normal liver cells and non‑cancerous tissues were compared at both transcriptional and translational levels. Correlation between SKA3 levels, clinicopathological characteristics and patient survival was also assessed. Gene set enrichment analysis(GSEA) was performed to identify SKA3‑associated pathways. Furthermore, SKA3 was knocked down and overexpressed in liver cancer cells, and then assessed the effect on cell proliferation, cell cycle, and tumor formation ability. Kaplan‑Meier survival analysis and log‑rank test were used to evaluate the association between SKA3 expression levels and prognosis. SKA3 mRNA and protein expression levels were significantly higher in liver cancer cell lines and clinical samples, compared with normal controls. Immunohistochemical analysis of 110patients revealed that upregulation of SKA3 correlated with clinical pathological characteristics and patient survival. GSEA showed that BENPORATH_PROLIFERATION gene set signaling pathways were correlated with SKA3 expression levels. Luciferase reporter activity assay revealed that knockdown of SKA3 significantly inhibited the activity of transcription factor E2F. Downregulation of SKA3 significantly inhibited cell proliferation, cell cycle arrest in G1‑Sphase and tumorigenesis both invitro and invivo, decreased the expression levels of cyclinD1 and phosphorylated‑-retinoblastoma and increased those of p21, suggesting a potential role of SKA3 in mediating tumor cell cycle and progression. SKA3 may function as an oncogene in liver cancer and may be a promising prognostic biomarker and candidate for targeted therapy.

Spindle and kinetochore‑associated complex subunit 3 accelerates breast cancer cell proliferation and invasion through the regulation of Akt/Wnt/β-catenin signaling.

Spindle and kinetochore‑associated complex subunit 3 (SKA3) has recently been identified as a novel regulator of carcinogenesis in multiple types of cancers. However, the function and potential regulatory mechanisms of SKA3 in breast cancer remain poorly understood. The present study was designed to gain a detailed relevance of SKA3 in breast cancer. Expression of SKA3 in breast cancer was examined via real-time quantitative PCR, western blotting and immunohistochemistry analysis. Malignant behaviors of breast cancer cells were investigated via cell counting kit-8, cell apoptosis, and transwell invasion assays. The activity of Wnt/β-catenin signaling was monitored via luciferase reporter assay. The tumorigenicity of breast cancer cells in vivo was assessed via xenograft tumor assay. SKA3 expression was elevated in breast cancer tissue and was correlated with shorter survival rates in breast cancer patients. Knockdown of SKA3 caused marked reductions in cellular proliferation and invasion in breast cancer cells, whereas SKA3 overexpression accelerated proliferation and invasion. Knockdown of SKA3 resulted in decreased Akt and glycogen synthase kinase-3β phosphorylation, and decreased expression of active β-catenin, which lead to the inactivation of Wnt/β-catenin signaling. Inhibition of Akt significantly reversed the SKA3 overexpression-induced activation of Wnt/β-catenin signaling. Inhibition of Wnt/β-catenin signaling markedly abrogated SKA3 overexpression-induced tumor-promotion effects, while re-activation of Wnt/β-catenin signaling significantly reversed SKA3 knockdown-mediated tumor-inhibition effects. Knockdown of SKA3 resulted in a significant decrease in breast cancer tumor formation in vivo. SKA3 accelerates proliferation and invasion in breast cancer through the modulation of Akt/Wnt/β-catenin signaling.

Identification of Spindle and Kinetochore-Associated Family Genes as Therapeutic Targets and Prognostic Biomarkers in Pancreas Ductal Adenocarcinoma Microenvironment.

AimThe role of spindle and kinetochore-associated (SKA) genes in tumorigenesis and cancer progression has been widely studied. However, so far, the oncogenic involvement of SKA family genes in pancreatic cancer and their prognostic potential remain unknown.MethodsHere, we carried out a meta-analysis of the differential expression of SKA genes in normal and tumor tissue. Univariate and multivariate survival analyses were done to evaluate the correlation between SKA family gene expression and pancreas ductal adenocarcinoma (PDAC) prognosis. Joint-effect and stratified survival analysis as well as nomogram analysis were used to estimate the prognostic value of genes. The underlying regulatory and biological mechanisms were identified by Gene set enrichment analysis. Interaction between SKA prognosis-related genes and immune cell infiltration was assessed using the Tumor Immune Estimation Resource tool.ResultsWe find that SKA1–3 are highly expressed in PDAC tissues relative to non-cancer tissues. Survival analysis revealed that high expression of SKA1 and SKA3 independently indicate poor prognosis but they are not associated with relapse-free survival. The prognostic value of SKA1 and SKA3 was further confirmed by the nomogram, joint-effect, and stratified survival analysis. Analysis of underlying mechanisms reveals that these genes influence cancer-related signaling pathways, kinases, miRNA, and E2F family genes. Notably, prognosis-related genes are inversely correlated with several immune cells infiltrating levels.ConclusionWe find that SKA1 and SKA3 expression correlates with prognosis and immune cell infiltration in PDAC, highlighting their potential as pancreatic cancer prognostic biomarkers.

SKA3 overexpression promotes cell proliferation and migration in breast cancer cell lines

Abstract Objectives Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide with a high mortality rate, despite early detection and treatment. Spindle and kinetochore-associated complex subunit 3 (SKA3) is closely correlated with patient outcomes in several cancers. The present study aimed to elucidate the role of SKA3 in BC. Methods The biological functions of SKA3 was investigated by proliferation and migration assays in MDA-MB-231 cells with stable SKA3 knockdown and Hs578T cells ectopically expressing SKA3. Gene Expression Omnibus datasets were utilised to determine the correlation between SKA3 expression and clinical features of BC patients. Results We confirmed that SKA3 mRNA expression is higher in breast tumour tissue than in normal tissue, and that higher SKA3 expression is associated with poor survival rate of BC patients. Knockdown of SKA3 reduced MDA-MB-231 cell proliferation and migration, whereas SKA3 overexpression enhanced the proliferative and migratory ability of Hs578T cells. We also found that SKA3 is involved in regulating cell cycle progression in mitotic exit. Conclusions These results suggest that SKA3 is correlated with BC cell proliferation and migration by promoting cell cycle progression, and could be a novel potential therapeutic target for BC treatment.

SKA3 Promotes Cell Growth in Breast Cancer by Inhibiting PLK-1 Protein Degradation.

Breast cancer (Bca) remains the most common form of malignancy affecting females in China, leading to significant reductions in the mental and physical health of those with this condition. While spindle and kinetochore associated complex subunit 3 (SKA3) is known to be linked with cervical cancer progression, whether it is similarly associated with Bca progression remains unknown. Using shRNA, we specifically knocked down the expression of SKA3 in Bca cell lines and then assessed the resultant changes in cell proliferation using CCK-8 and colony formation assays. In addition, we used western blotting to quantify the expression levels of relevant proteins in these cells, and we assessed the interaction between SKA3 and polo-like kinase-1 (PLK-1) via co-immunoprecipitation.In this study, we observed elevated SKA3 expression in Bca tissues and cell lines. When we knocked down SKA3 expression in Bca cells, we were able to determine that it functions in an oncogenic manner so as to promote the growth and proliferation of these cells in vitro. From a mechanistic perspective, we were able to show that in Bca cells SKA functions at least in part via interacting with PLK-1 and preventing its degradation. In summary, we found that SKA3 is able to regulate PLK-1 degradation in Bca cells, thus controlling their growth and proliferation. These results highlight SKA3 as a potentially viable target for anti-cancer drug development aimed at combatting Bca.

Prognostic value and functional bioinformatic analysis of spindle- and kinetochore-associated protein 1 in stage IIA esophageal squamous cell carcinoma.

As one of the most common malignant tumors of the digestive tract, esophageal squamous cell carcinoma (ESCC) is an advanced metastatic cancer with an extremely high mortality rate and the highest prevalence rate in China. Spindle- and kinetochore-associated protein 1 (SKA1), an essential member involved in chromosome separation during mitosis, has been indicated as a potential biomarker in the pathogenesis and development of various types of malignant tumors; however, the exact functions of SKA1 in ESCC are still unclear. SKA1 expression was explored in stage IIA ESCC and corresponding healthy esophageal mucosa tissues through immunohistochemistry and reverse transcription-quantitative polymerase chain reaction and was further validated using The Cancer Genome Atlas (TCGA) database of the online tool UALCAN. Then, the clinicopathological correlations of SKA1 were analyzed based on the follow-up data. Furthermore, using the online tool LinkedOmics, the correlation test, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of SKA1 were analyzed using high-throughput sequencing data of ESCC patients from TCGA dataset. The expression level of SKA1 was markedly upregulated in ESCC tissues. Upregulation of SKA1 significantly correlated with higher pathological T stage (P = 0.003) and poorer overall survival (P = 0.013). GO and pathway enrichment analyses of SKA1 in ESCC revealed that SKA1 was involved in a number of classical cell cycle-related pathways that contribute to special biological processes in tumorigenesis and development of ESCC. The results of this study demonstrate that SKA1 may act as a prognostic biomarker for stage IIA ESCC. Combined with the bioinformatic analysis, SKA1 could potentially serve as a therapeutic target for ESCC. The results coming from the present study demonstrated that SKA1 may act as a prognostic biomarker for stage IIA ESCC. Combined with the bioinformatic analysis, SKA1 could serve as a potential therapeutic target for ESCC.

SKA1 promotes malignant phenotype and progression of glioma via multiple signaling pathways

BackgroundSpindle and kinetochore associated protein 1 (SKA1) is a protein involved in chromosome congression and mitosis. It has been found to be upregulated and oncogenic in several human cancers. Herein, we investigated the precise role of SKA1 in the progression and malignant phenotype of human glioma.MethodsBioinformatic analysis was carried out based on the RNA-seq data and corresponding clinical data from GEO, TCGA and CGGA databases. Western blot was performed to analyze the expression of SKA1 in clinical samples and signaling pathway proteins in glioma cells, respectively. CCK8 assay, colony forming assay and EdU assay were performed to assess the cell viability. Cell migration and invasion assays were also performed. Moreover, xenograft model was established and the expression of SKA1 was assessed in the xenograft by immunohistochemistry.ResultsSKA1 expression is positively correlated with glioma grade and could be a promising biomarker for GBM. Moreover, overexpression of SKA1 may lead to poor prognosis in glioma. Downregulation of SKA1 attenuated cell viability, migration, and invasion in U251, U87, LN229 and T98 cells. Furthermore, GSEA analysis demonstrated that SKA1 was involved in the cell cycle, EMT pathway as well as Wnt/β-catenin signaling pathway, which were then confirmed with Western blot analysis.ConclusionSKA1 promotes malignant phenotype and progression of glioma via multiple pathways, including cell cycle, EMT, Wnt/β-catenin signaling pathway. Therefore, SKA1 could be a promising therapeutic target for the treatment of human gliomas.

SKA3 Promotes tumor growth by regulating CDK2/P53 phosphorylation in hepatocellular carcinoma

Spindle and kinetochore-related complex subunit 3 (SKA3) is a component of the spindle and kinetochore-related complexes and is essential for accurate timing of late mitosis. However, the relationship between SKA3 and hepatocellular carcinoma (HCC) has not yet been fully elucidated. Gene expression omnibus (GEO) (GSE62232, GSE45436, GSE6764, and GSE36376) and The Cancer Atlas (TCGA) datasets were analyzed to identify differential expression genes. Cell proliferation ability was analyzed using Cell Counting Kit-8 (CCK8) assay and plate clone formation assay, while scratch wound healing assay and transwell assay were used to analyze cell invasion. The role of SKA3 in vivo was explored using subcutaneous xenotransplantation model and lung metastasis model. Bioinformatics analysis found that hepatocellular carcinoma patients with high levels of expression of SKA3 have a poor prognosis. Similarly, immunohistochemical staining of 236 samples of tumors also found higher SKA3 expression in them, than in adjacent normal liver tissues. Significant levels of inhibition of in vivo and in vitro tumor proliferation and invasion result from the downregulation of SKA3. Mechanistically, SKA3 was found to affect tumor progression through the cell cycle and P53 signaling pathway as shown by the gene enrichment analysis (GSEA). G2/M phase arrest and severe apoptosis was also found to result from SKA3 knockdown, as shown by the inhibition of CDK2/p53 phosphorylation together with downregulation of BAX/Bcl-2 expression in HCC cells. Overall, these findings uncover the role of SKA3 in regulating the apoptosis and proliferation of hepatocellular carcinoma cells. This study was able to uncover new information on the tumorigenesis mechanism in hepatocellular carcinoma.

Computational assessment of SKA1 as a potential cancer biomarker

Abstract Background Spindle and kinetochore associated complex subunit 1 (SKA1) is an essential component of SKA complex, which is required for the proper formation of kinetochore–microtubule attachment and timely mitotic progression. The aim of this study is to perform detailed analyses of SKA1 genomic and expression alterations in cancers and to assess SKA1 as a biomarker for predicting human cancers and patient prognosis. Materials and methods Missense mutations from human cancers were extracted, deleterious missense mutations were predicted and shown on 3D SKA1 protein. SKA1 expression and the effect of SKA1 expression on patient survival were investigated in human cancers. Results and discussion Most of the predicted deleterious mutations were detected on microtubule-binding domain of SKA1, suggesting mutations on microtubule-binding domain might be more relevant in human cancers. High SKA1 expression was detected in various cancers. In addition, patients with high SKA1 expression showed poor overall survival compared to patients with low SKA1 expression in breast, lung and gastric cancers. Conclusion These results suggest that high SKA1 expression might be a prognostic and predictive biomarker for several cancers and mainly mutations in the microtubule-binding domain of SKA1 might have a deleterious effect for SKA1.

LINC00339 promotes growth and invasiveness of hepatocellular carcinoma by the miR-1182/SKA1 pathway.

Background: Extensive research has shown that long noncoding RNA (lncRNA) is involved in tumorigenesis, including hepatocellular carcinoma (HCC). The lncRNA LINC00339 was reported to regulate the development of lung cancer or breast cancer. However, whether LINC00339 participates in HCC progression remains unclear. Here, our results showed that LINC00339 was upregulated in HCC.Methods: qRT-PCR and in situ hybridization (ISH) was used to analyze LINC00339 expression in tumor tissues and cell lines. CCK8 and colony formation assays were used to analyze cell proliferation. Transwell assay was used to analyze cell migration and invasion. Xenograft experiment was used to test tumor growth in vivo.Results: LINC00339 overexpression was correlated with an advanced stage, metastasis, and bad prognosis in HCC patients. Functional investigation showed that LINC00339 knockdown significantly suppressed HCC cell proliferation, migration, and invasion. Moreover, decreased LINC00339 expression inhibited HCC growth in vivo. Mechanistically, LINC00339 could interact with miR-1182 to promote SKA1 expression. We also demonstrated that SKA1 acted as an oncogene and SKA1 upregulation reversed the effect of LINC00339 silencing.Conclusion: Our results illustrated that the LINC00339/miR-1182/SKA1 axis plays an essential role in HCC progression.

PPARγ promotes diabetes-associated centrosome amplification via increasing the expression of SKA1 directly at the transcriptional level.

We haverecently published that type 2 diabetes can induce cell centrosome amplification due to the action of high glucose, palmitic acid, and insulin, and ROCK1 and 14-3-3σ are signal mediators. In this study, we further investigated the molecular mechanisms of the centrosome amplification in colon cancer HCT116 cells. Treatment of the cells with high glucose, palmitic acid, and insulin increased the expression of peroxisome proliferator-activated receptor γ (PPARγ) as well as the spindle and kinetochore associated protein 1 (SKA1), knockdown of each of which resulted in the inhibition of the treatment-triggered centrosome amplification. Knockdown of PPARγ inhibited the treatment-evoked increase in the SKA1 level, whereas knockdown of SKA1 did not modify the treatment-increased PPARγ level. We found a predicted binding site for PPARγ in the promoter region of the SKA1 gene from the JASPAR database. Experimental results showed that the treatment increased the messenger RNA level of SKA1, which could be inhibited by PPARγ chemical inhibitor or small interfering RNA. Moreover, we were able to show that PPARγ could bind to the binding site in the SKA1 gene promoter, which was increased by the experimental treatment. In conclusion, it is suggested that the pathophysiological factors in type 2 diabetes, high glucose, palmitic acid, and insulin, induce the cell centrosome amplification through the PPARγ-SKA1 pathway, in which PPARγ increases the expression of SKA1 via directly enhancing the SKA1 gene transcription.

SKA1 induces denovo MTX-resistance in osteosarcoma through inhibiting FPGS transcription.

Denovo methotrexate (MTX)-resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the denovo MTX-resistant OS cell line SF-86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to denovo MTX-resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with denovo MTX-resistance and poor 5-year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly-γ-glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX-PG. We further demonstrated that SKA1 interacts with DNA-directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX-sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF-86 cells due to the formation of an inhibitory SKA1-RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF-86. Collectively, our study has established the denovo MTX-resistant cell line SF-86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of denovo MTX-resistance in OS.

SKA1 overexpression is associated with poor prognosis in hepatocellular carcinoma

BackgroundSKA1, an important mitosis protein, has been indicated in the initiation and progression of several malignancies. However, its clinical significance in hepatocellular carcinoma (HCC) remain to be elucidated.MethodsmRNA expression of SKA1 was examined in 126 HCC and paired non-neoplastic tissues using real-time PCR and validated in The Cancer Genome Atlas (TCGA) database. SKA1 protein expression was detected using immunohistochemistry in the 126 HCC tissues and its associations with clinicopathological parameters and prognosis were analyzed. Hierarchical cluster analysis and gene set enrichment analysis (GSEA) were performed in selected Gene Expression Omnibus data sets.ResultsSKA1 mRNA expression was significantly elevated in HCC tissues from both local hospital and TCGA database. Immunohistochemistry revealed that increased SKA1 expression was present in 65 of the 126 cases and was significantly associated with higher serum alpha-fetoprotein concentration, larger tumor size and higher TNM stage. Patients with positive SKA1 expression showed significantly worse overall and relapse-free survival. Multivariate Cox regression analysis revealed that SKA1 was an independent predictor of patient prognosis. Gene expression profiling analysis of public data showed that high-SKA1 expression HCC tissues had similar gene expression profiles with fetal liver tissues. Moreover, GSEA showed that genes up-regulated in high SKA1 HCC subgroup were significantly enriched in cell cycle pathway, while genes down-regulated were significantly enriched in apoptosis pathway.ConclusionsOur findings indicate that the oncofetal gene SKA1 might be involved in the progression of the HCC and could serve as a prognostic marker for HCC.

SKA3 promotes cell proliferation and migration in cervical cancer by activating the PI3K/Akt signaling pathway

BackgroundCervical cancer (CC) is one of the most common cancers among females worldwide. Spindle and kinetochore-associated complex subunit 3 (SKA3), located on chromosome 13q, was identified as a novel gene involved in promoting malignant transformation in cancers. However, the function and underlying mechanisms of SKA3 in CC remain unknown. Using the Oncomine database, we found that expression of SKA3 mRNA is higher in CC tissues than in normal tissues and is linked with poor prognosis.MethodsIn our study, immunohistochemistry showed increased expression of SKA3 in CC tissues. The effect of SKA3 on cell proliferation and migration was evaluated by CCK8, clone formation, Transwell and wound-healing assays in HeLa and SiHa cells with stable SKA3 overexpression and knockdown. In addition, we established a xenograft tumor model in vivo.ResultsSKA3 overexpression promoted cell proliferation and migration and accelerated tumor growth. We further identified that SKA3 is involved in regulating cell cycle progression and the PI3K/Akt signaling pathway via RNA-sequencing (RNA-Seq) and gene set enrichment analyses. Western blotting results revealed that SKA3 overexpression increased levels of p-Akt, cyclin E2, CDK2, cyclin D1, CDK4, E2F1 and p-Rb in HeLa cells. Additionally, the use of an Akt inhibitor (GSK690693) significantly reversed the cell proliferation capacity induced by SKA3 overexpression in HeLa cells.ConclusionsWe suggest that SKA3 overexpression contributes to CC cell growth and migration by promoting cell cycle progression and activating the PI3K–Akt signaling pathway, which may provide potential novel therapeutic targets for CC treatment.

Abstract 2526: MicroRNA expression signature of patients with tyrosine kinase inhibitors failure: miR-10a-5p inhibits cancer cell aggressiveness in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is a disease in which cells undergo oncogenic transformation in the kidney tubules. The five-year survival rate of advanced stage RCC is poor (5-10%) due to recurrence or distant metastasis. Recently, anti-angiogenic multi-tyrosine kinase inhibitors (TKIs) have been developed and have been used as first and second line treatments for RCC. However, these treatments extend progression-free survival only slightly, and relapse and metastasis eventually develop in most patients. The molecular mechanisms of RCC recurrence, metastasis and drug resistance are not yet fully understood. Therefore, analysis of the molecular mechanisms underlying RCC development and progression and studies of novel oncogenic pathways based on current genome-based approaches could significantly improve diagnosis, therapy, and prevention of the disease. miRNAs (miRNAs) are small noncoding RNAs that function to fine tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-depending manner. The unique characteristic function of miRNAs is to regulate RNA transcripts in human cells. Therefore, dysregulated expression of miRNAs can disrupt tightly regulated RNA networks in cancer cells. In this study, we constructed a miRNA expression signature to identify pathways activated by TKI treatment using autopsy specimens from patients with RCC. We have sequentially identified tumor-suppressive miRNA and its regulated RCC pathways based on the signature. The aim of this study was to investigate the functional significance of miR-10a-5p and to identify the molecular targets and pathways mediated by miR-10a-5p in RCC cells. The expression levels of miR-10a-5p were significantly reduced in RCC clinical specimens and RCC cell lines compared with non-cancerous kidney tissues (P < 0.001). TCGA data showed that the overall survival of low miR-10a-5p expression group was significantly shorter than that of high expression group (P = 0.00408). Restoration of miR-10a-5p significantly inhibited cancer cell migration and invasion in RCC cell lines (P < 0.0001). Spindle and kinetochore associated complex subunit 1 (SKA1) was identified as a direct target gene of miR-10a-5p by genome-wide gene expression analysis and in silico analysis. Overexpression of SKA1 was observed in RCC clinical specimens. Moreover, the overall survival of high SKA1 expression group was significantly shorter than that of low expression group by TCGA analysis (P = 1.44E-07). Tumor-suppressive miR-10a-5p was identified by using miRNA signature of patients with TKI failure. Overexpression of SKA1 might be involved in RCC aggressiveness, metastasis and drug resistance. Elucidation of tumor-suppressive miRNAs regulated molecular pathways and targets could provide new information on potential therapeutic strategies in the disease. Citation Format: Takayuki Arai, Atsushi Okato, Akira Kurozumi, Mayuko Kato, Yusuke Goto, Keiichi Koshizuka, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. MicroRNA expression signature of patients with tyrosine kinase inhibitors failure: miR-10a-5p inhibits cancer cell aggressiveness in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2017-2526